The advent of highly active antiretroviral therapy (HAART), which involves the use of three or more antiretroviral drugs, has led to a significant improvement in the care and survival of patients infected with HIV-1. In patients not infected with resistant strains of the virus, HAART typically results in a dramatic decrease in viral load often from levels of 10,000-100,000 RNA copies/ml of plasma to less than 50 copies/ml (3).
Given the dramatic effects of HAART, it was proposed that complete elimination of the virus might be possible within 2 to 3 years (36). However, even after long-term suppression of viral replication with HAART, the virus rapidly rebounds after therapy is discontinued (7,12). A key contributor to viral rebound appears to be a reservoir of latently infected cells, including CD4+ memory T cells. The half-life of the latently infected population is quite long, and it is estimated that it would take over 60 years of HAART to eliminate this population (15). Therefore, life-long HAART would be required to control infection in patients.
Retroviruses, including HIV-1, are RNA viruses that replicate through a DNA intermediate and integrate very efficiently into the genome of an infected cell forming a provirus. Once the provirus is formed, it is maintained in the genome of the infected cell and transferred to daughter cells in the same fashion as any other genetic element within the cellular genome. Thus, the virus has the potential to persist if it infects long-lived cells such as memory T cells. It has been known since 1986 (17) that HIV-1 can establish a latent infection in culture. It was found that a human T cell line infected with replication-competent virus could develop a latent infection in which the provirus was dormant but could be reactivated upon stimulation. Since then it has been established that a number of cytokines including tumor necrosis factor (TNF)-α and even a small molecule such as the phorbol ester, phorbol 12-myristate 13-acetate (PMA) can reactivate latent proviruses (30).
The role that latency is playing in preventing clearance of the virus infection has become evident in recent years. Patients that had been successfully treated with HAART in which viral RNA was maintained at levels below 50 copies/ml in the plasma for years, experienced rapid virus rebound upon withdrawal of therapy (7,12). Moreover, it was found that after T cell activation, virus could be isolated from CD4+ T cells taken from these patients making it clear that to eradicate the virus it will be necessary to eliminate the latently infected cells (10,16,19,45).
There have been attempts to flush the latent virus from infected individuals by non-specific activation of T cells to “turn on” latent proviruses. As part of this approach, the patients remain on HAART to prevent new infections, and the infected cells from which the latent proviruses are activated should die due to cytotoxic effects of viral expression and/or because of targeting by the immune system which can recognize the cells once they begin to express the viral proteins (3). One approach employed the combination of a monoclonal antibody against CD3 on T cells plus IL-2 to activate T cells and consequently the latent proviruses (37). Other approaches have used IL-2 with or without additional cytokines (8,13,31,41). To date, none of these protocols have been successful, and at least some of them have toxic side effects, which is not surprising considering the massive T cell activation that occurs. One plausible reason for the lack of latent provirus clearance could be due to the inability of the therapeutic regime to reach all of the latent reservoirs.
A potentially fruitful approach to eliminating virus infection would be to identify small molecules with pharmacological properties that allow these molecules to reach hard to access latent reservoirs in order to activate latent proviruses. There is precedent for a small molecule that can activate latent HIV-1 proviruses, since it was found that the tumor-promoting phorbol ester PMA could stimulate latent virus. This has led to recent studies with a non-tumor promoting phorbol ester, prostratin, which has also been found to be able to activate latent virus leading to the hypothesis that prostratin can be employed to help eradicate latent infection (2729). However, it is not presently known whether prostratin has the appropriate pharmacological properties to enable total clearance of latent virus nor is it certain that only one drug will be enough for latent virus elimination. Moreover, it was recently reported that prostratin displayed significant cytotoxicity putting into question its use in a clinical setting (43).
Thus, there is a need in the art for further strategies to discover new drugs capable of activating latent HIV-1. Preferably, there is a need for a cell-based assay that can be utilized in high throughput screening (HTS) to discover novel compounds capable of activating latent HIV-1.